NOVEL TRANSPOSONS AND PLASTICITY ZONES OF HELICOBACTER PYLORI

描述（由申请人提供）： 转座因子（TE）对基因组组织和进化、调控、发育编程以及人类健康和疾病有重要贡献-最著名的是细菌耐药性转座子、致癌逆转录病毒和V（D）J免疫系统重组。TE在运动机制和调节方面是多样的，它们的分析为蛋白质-核酸相互作用和细胞调节机制提供了有价值的见解。病原体中的TE通常含有影响表型（如毒力）的辅助基因。这一提议源于我们在胃病原体幽门螺杆菌中发现了新的“可塑区”转座子（TnPZs）。这些TE编码酪氨酸重组酶家族的XerC/XerD分支的新成员（“XerT”）、IV型分泌系统（“tfs 3”）和含有显著的DNA甲基化酶和RNA解旋酶基序的大蛋白（OrfQ）（2800 - 4000个残基，取决于菌株）。OrfQ样蛋白在其他不相关的细菌病原体的基因组序列中是明显的。我们假设（i）TnPZ是接合转座子，通过其Tfs 3蛋白复合物在细菌细胞之间传递，并使用XerT蛋白插入新位点;（ii）推定的DNA甲基化酶OrfQ可能导致靶组织中的表观遗传（DNA修饰）变化，可能影响胃病理学和疾病。在具体目标1中，我们将研究H. pylori和E.杆菌自原型（E. coli）的XerC/XerD蛋白只对一个染色体序列具有特异性，而TnPZs则可插入多个位点，因此对XerT作用的研究有助于加深对蛋白质-核酸特异性及其进化的理解。在具体目标2中，我们将在感染的哺乳动物细胞和细菌细胞中检测OrfQ介导的DNA甲基化。OrfQ的结构域结构表明，对其作用的研究可以为慢性感染期间细菌-宿主相互作用的动力学提供新的见解。总之，这些R21研究的结果应加强对转座相关现象以及感染和毒力机制的理解，并提供支持未来几年预期更大规模的RO 1型应用所需的数据。 公共卫生相关性： 从转座子TnPZ研究中获得的基本见解将丰富对微生物病原体进化和人类疾病的理解。特别是对OrfQ的研究，可以揭示细菌-宿主相互作用的新维度和疾病病理学中重要的人类表观遗传变化的起源，并改善感染和相关病理学（包括胃癌）的诊断和治疗。

DESCRIPTION (provided by applicant): Transposable elements (TEs) contribute importantly to genome organization and evolution, regulation, developmental programming, and human health and disease -- most famously, as bacterial drug resistance transposons, oncogenic retroviruses, and V(D)J immune system recombination. TEs are diverse in mechanisms and regulation of movement, and their analyses provide valuable insights into protein-nucleic acid interactions and cellular regulatory mechanisms. TEs in pathogens often contain auxiliary genes that affect phenotypes such as virulence. This proposal stems from our discovery of novel "plasticity zone" transposons (TnPZs) in the gastric pathogen Helicobacter pylori. These TEs encode a novel member of the XerC/XerD branch of the tyrosine recombinase family ("XerT"), a type IV secretion system ("tfs3"), and a large protein (OrfQ) (2800 - 4000 residues, depending on strain) that contains prominent DNA methylase and RNA helicase motifs. OrfQ-like proteins are evident in genome sequences of other unrelated bacterial pathogens. We hypothesize that (i) TnPZs are conjugative transposons, passed between bacterial cells via their Tfs3 protein complex, and inserted into new sites using XerT protein; and (ii) that the putative DNA methylase OrfQ may cause epigenetic (DNA modification) changes in target tissues that could impact on gastric pathology and disease. In Specific Aim 1 we will study the mechanism and control of TnPZ excision and transposition in H. pylori and E. coli. Since prototype (E. coli) XerC/XerD proteins are specific for one unique chromosomal sequence whereas TnPZs insert into many sites, studies of XerT action should enhance understanding of protein-nucleic acid specificity and its evolution. In Specific Aim 2 we will test for OrfQ-mediated DNA methylation in infected mammalian cells and in bacterial cells. OrfQ's domain structure suggests that studies of its action could give new insights into the dynamics of bacterial-host interactions during chronic infection. In conclusion, results of these R21 studies should enhance understanding of transposition-related phenomena, and of infection and virulence mechanisms, and also provide data needed to support an anticipated larger RO1-type application in coming years. PUBLIC HEALTH RELEVANCE: The fundamental insights to be gained from transposon TnPZ studies will enrich understanding of microbial pathogen evolution and human disease. Studies of OrfQ, in particular, could reveal a new dimension of bacterial-host interactions and the origin of human epigenetic changes important in disease pathology, and result in improved diagnosis and therapy for infections and associated pathologies, including gastric cancer.